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| <font color=" #990000" face="arial" size="6"> | | <font color=" #990000" face="arial" size="6"> |
- | <br> | + | <br><br> |
- | <b>Plug `n´ Play with DNA – making molecular biology easier</b><br> <br> | + | <b>Plug 'n' Play with DNA assembly standard</b><br> <br> |
| </font> | | </font> |
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| | | |
- | <p align="justify"> | + | |
- | We imagine that iGEM and synthetic biology should be about assembling BioBricks fast. It should be easy to combine any thinkable part, device or existing BioBrick. Unfortunately, classical cloning techniques can cause problems and even PCR can be cumbersome, if you have little or no laboratory experience. <br> <br>
| + | |
- | Therefore, we introduce a simple and fast way of building new devices, vectors or whatever preferred with ready to use PCR products. All what is needed is to select the favourite BioBricks and one of the ready to use destination vector, and mix it with USER enzyme.The assembled vector is ready for transformation in competent <i>E. coli</i> cells only 70 minutes later.<br>
| + | <table cellpadding=10px> |
- | No need to worry if the destination vector has been fully linearized, no need to perform site-directed mutagenesis to remove unwished restriction sites, just simple, fast, and easy.</p>
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| + | <td width="500px" height="100%" valign="top" valign="center" > |
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- | <font color=" #990000" face="arial" size="5"> | + | |
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| + | <!--<span id="test">test</span>--> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#Making molecular biology easier" class="h1"> Making molecular biology easier</a><br><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#How it works" class="h1"> How it works</a><br><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#The Procedure of assembly" class="h1"> The procedure of assembly</a><br><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#Plug 'n' Play benefits" class="h1"> Plug 'n' Play benefits</a><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#Improvements of BBF RFC 39" |
| + | class="h2"> Improvements of BBF RFC 39</a><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#What you get" class="h2"> What you get</a><br><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#The design of the system" class="h1"> The design of the system</a><br><br> |
| + | <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly#References" class="h1">References</a><br><br> |
| + | |
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| + | </div> |
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| + | <td width="75%" height="100%" valign="top"> |
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| <br> | | <br> |
- | <b>The new assembly system </b><br> | + | |
- | </font> | + | <a name="Making molecular biology easier"></a><h1><b>Making molecular biology easier</b></h1> |
| + | <p align="justify"> |
| + | We imagine that iGEM should be about fast assembly of BioBricks, leaving more time for the actual project. It should be easy to combine any thinkable part, device or existing BioBrick. Unfortunately, classical cloning techniques can cause problems and even PCR can be cumbersome, if you have little or no laboratory experience. <br> <br> |
| + | Therefore, we introduce a simple and fast way of building new devices and vectors with ready to use PCR products. All that is needed is to select the favorite BioBricks and one of the ready to use backbone vectors, and mix it with USER enzyme. The assembled vector is ready for chemical transformation into competent <i>E. coli</i> cells only 70 minutes later.The 8-9 nucleotide overhangs (linkers) flanking the parts, can hybridize to complementary overhangs without the use of DNA-ligases.<br> |
| + | No need to worry whether the destination vector has been fully linearized, no need to perform site-directed mutagenesis to remove unwished restriction recognition sites - just simple, fast, and easy.</p> |
| + | <br> |
| + | |
| <p align="justify"> | | <p align="justify"> |
- | Successful assembly of up to six biological parts in one reaction is possible, reducing the plasmid construction time significantly. The upper limit of fragments that efficiently can be assembled has not been delineated (1). Due to the 8-9 basepair overhangs the parts are ligated without the use of DNA-ligases.<br>
| + | It is possible to successfully assemble six biological parts in one reaction, reducing the plasmid construction time significantly. The upper limit of fragments that efficiently can be assembled has not yet been delineated (1). <br> |
- | In 2009 the first team from DTU to participate in iGEM introduced the USER fusion Assembly standard <a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_39:_The_USER_cloning_standard">[BBF RFC 39]</a>. This year we introduce a more standardized version allowing easier use of the system. The Plug `n´ Play assembly standard can be found <a href="http://bbf.openwetware.org/RFC.html#BBF_RFC_80:_Plug_.27n.27_Play_with_DNA"> here</a> (BBF RFC 80). | + | In 2009 the first team from DTU participated in iGEM and introduced the USER fusion Assembly standard <a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_39:_The_USER_cloning_standard">[BBF RFC 39]</a>. This year we introduce a standardized version allowing easier use of the system. The Plug 'n' Play assembly standard can be found <a href="http://bbf.openwetware.org/RFC.html#BBF_RFC_80:_Plug_.27n.27_Play_with_DNA"> here</a> (BBF RFC 80). |
| </p><br> | | </p><br> |
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- | <font color="#990000" face="arial" size="4" >
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- | <p align="left">
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- | <b></b>The Plug `n´ Play assembly - How it works
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- | </p>
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- | </font>
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| + | <a name="How it works"></a><h2><b>How it works</b></h2> |
| <p align="justify"> | | <p align="justify"> |
- | We have demonstrated successful assembly of up to six biological parts in one reaction is possible e.g. <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678068"> (BBa_K678068)</a>. | + | We have demonstrated that successful assembly of up to six biological parts in one reaction is possible e.g. <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678068"> (BBa_K678068)</a>. |
- | The entire process of combining 6 BioBricks are illustrated in the figure below. The number of BioBricks can be adjusted according to preferred as well as introducing mutation see <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization"> Customization</a>. </p><br> | + | The entire process of combining six BioBricks is illustrated in the figure below. The number of BioBricks can be adjusted as preferred and for instance mutations can easily be introduced by <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization">customizing</a> the standard. </p><br> |
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- | <font color="#990000" face="arial" size="4" >
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- | <p align="left">
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- | <b></b>The Procedure of assembly <br>
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- | </p>
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- | </font>
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- | Please refer to our Biobrick Assembly Standard <a href="http://bbf.openwetware.org/RFC.html#BBF_RFC_80:_Plug_.27n.27_Play_with_DNA">BBF RFC 80</a> for a detailed protocol.<br> | + | <a name="The Procedure of assembly"></a><h2><b>The procedure of assembly</b></h2> |
- | 1. Pre-produced PCR products are picked from the Plug `n´ Play kit.<br>
| + | Please refer to our Plug 'n' Play with DNA assembly standard <a href="http://bbf.openwetware.org<br>/RFC.html#BBF_RFC_80:_Plug_.27n.27_Play_with_DNA">BBF RFC 80</a> for a detailed protocol.<br><br> |
- | 2. USER enzyme mix and buffer is added. This will remove the uracil, making sticky end overhangs on all BioBricks. All BioBricks and backbone plasmid have matching sticky ends, and the BioBricks will self-assemble in the plasmid. <br>
| + | <dt> |
- | 3. Transformation in <i>E. coli</i>. Reduced incubation of the transformation mix is obtained when using ampicillin as antibiotic resistance marker. <br>
| + | <dd><li>Pre-produced PCR products are selected from the Plug 'n' Play kit.</li></dd> |
| + | <dd><li> USER enzyme mix and buffer is added. This will remove the uracils, resulting in sticky ends on all BioBricks. The BioBricks and the backbone plasmid have matching sticky ends, and will self-assemble. </li></dd> |
| + | <dd><li> Chemical transformation of <i>E. coli</i>. The time of the final incubation of the transformation mix can be reduced to 30 min when using ampicillin as antibiotic resistance marker and still results in a high number of transformants. |
| | | |
- | If a part is not incluede in the Plug `n´ Play kit a simple PCR amplification can performed on the part or BioBricks intended for the assembly. The PCR product should subsequently be gel band purified. | + | |
| + | If a part is not included in the Plug 'n' Play kit a simple PCR amplification can be performed on the part intended for the assembly in accordance to the <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization">Customization</a>.</li></dd> |
| + | </dt> |
| <br><br> | | <br><br> |
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- | <font color="#990000" face="arial" size="4"> | + | <a name="Plug 'n' Play benefits"></a><h2><b>Plug 'n' Play benefits</b></h2> |
- | <p align="left"> | + | |
- | <b></b>Plug `n´ Play benefits <br> | + | |
- | </p> | + | |
- | </font>
| + | |
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| <p align="justify"> | | <p align="justify"> |
| | | |
- | <b>Improvements of BBF RFC 39</b> <br> | + | <a name="Improvements of BBF RFC 39"></a><h3><b>Improvements of BBF RFC 39</b></h3> |
- | • No need to use a USER cassette, thereby completely avoiding the use of restriction enzymes. <br>
| + | <dt> |
- | • Standardized linkers <br> <br>
| + | <dd><li>No need to use a USER cassette, thereby completely avoiding the use of restriction enzymes and increasing the flexibility of the system. </li></dd> |
| + | <dd><li>Standardized linkers </li> </dd> |
| + | </dt> |
| + | <p align="justify"> |
| | | |
- | <b>What you get</b> <br> | + | <a name="What you get"></a><h3><b>What you get</b></h3> |
- | • Rapid assembly of biological parts <br>
| + | <dt> |
- | • A well documented system <br>
| + | <dd><li>Rapid assembly of biological parts </li></dd> |
- | • Assembly of multiple biobricks in one step <br>
| + | <dd><li>A well documented system </li></dd> |
- | • No illegal restrictionsites <br>
| + | <dd><li>Assembly of multiple biobricks in one step </li></dd> |
- | • 100% restriction enzyme free <br>
| + | <dd><li>No illegal restriction sites </li></dd> |
- | • 100% DNA ligase free <br>
| + | <dd><li>100% restriction enzyme free </li></dd> |
- | • High fidelity ensured by PfuTurbo® 7x Hotstart DNA polymerase <br>
| + | <dd><li>100% DNA ligase free </li></dd> |
- | • Directionality of inserts are supported <br>
| + | <dd><li>High fidelity ensured by PfuTurbo® 7x Hotstart DNA polymerase </li></dd> |
- | • Any vector can be made Plug’n’Play compatible <br>
| + | <dd><li>Directionality of inserts are supported </li></dd> |
- | • Suitable for large scale high throughput projects <br><br>
| + | <dd><li>Any vector can be made Plug’n’Play compatible </li></dd> |
| + | <dd><li>Suitable for large scale high throughput projects </li></dd> |
| + | </dt> |
| </p> | | </p> |
- | | + | <br><br> |
| <p align="justify"> | | <p align="justify"> |
- | Vectors for Plug `n´ Play have been prepared by PCR ensuring 100% linear fragments. This means that the occurrences of false positives due to the presence uncut vector can be completely avoided (2).<br><br> | + | Vectors for Plug 'n' Play have been prepared by PCR ensuring 100% linear fragments. This means that the occurrence of false positives after bacterial transformation can be completely avoided (2).<br><br> |
| </p> | | </p> |
| | | |
| <p align="justify"> | | <p align="justify"> |
- | Drawbacks of the USER cassette (2):<br>
| |
- | • The USER cassette has to be inserted into the vector of choice, which has to be done by classical cloning.<br>
| |
- | • Fragments can only be inserted at the site of the USER cassette making it inflexible<br>
| |
- | • The USER cassette has to be digested by a restriction endonuclease and a nicking enzyme<br><br>
| |
- | </p>
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- |
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- |
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| The USER compatible backbone vectors were made by PCR amplification with primers containing the standardized linkers 6 and 1. | | The USER compatible backbone vectors were made by PCR amplification with primers containing the standardized linkers 6 and 1. |
- | For all the PCR reactions it is important to remove the template vector, to insure assembly in USER reaction and avoid inauspicious transformation and false positive. The template can simply be removed by DpnI treatment (2). However, a gel band purification was instead performed, when preparing the vector to further insure a high degree a purification of our BioBricks.<br><br> | + | For all the PCR reactions it is important to remove the template vector before utilizing it in the USER reaction to avoid inauspicious transformations and false positives. The template can be removed by DpnI treatment or by gel band purification(2). </p><br> |
| | | |
- | Previous studies have demonstrated a high level of correct assembly and directionality of fragments (2,3) and although we did not make such analysis ourselves it was in general our perception that this was the case, of the more than 25 different Plug `n´ Play assemblies we performed we encountered no more than 2 false-positives.<br><br> | + | <p align="justify"> |
| + | Previous studies have demonstrated a high level of correct assembly and directionality of fragments (2,3) and although we did not make such analysis ourselves it was in general our perception that this was the case, as of the more than 25 different Plug 'n' Play assemblies performed we encountered no more than two false-positives.</p><br> |
| | | |
- | It is well known that mutations can be introduced during PCR. To minimize mutations the X7 proof-reading polymerases (4) are used which are fully compatible with the Plug `n´ Play standard. The x7 proof-reading polymerase helps lowering the risk of introducing mutations. The concern for introduction mutations is specially regarding the gene of interest (GOI), because mutations occurring in the vector backbone or resistance cassette would in worst case scenario mean the vector might not be able to propagate. | + | <p align="justify"> |
| + | It is well known that mutations can be introduced during PCR. To minimize mutations the x7 proof-reading polymerase (4) that are fully compatible with the Plug 'n' Play standard should be used. The x7 proof-reading polymerase helps lowering the risk of introducing mutations.</p> |
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- | <br>
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| <br> | | <br> |
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- | <font color="#990000" face="arial" size="5"> | + | <a name="The Design of The System"></a><h2><b>The design of the system</b></h2> |
- | <p align="left"> | + | |
- | <b></b>The Design of The System <br> | + | |
- | </p> | + | |
- | </font> | + | |
- | <br>
| + | |
- | | + | |
| <p align="justify"> | | <p align="justify"> |
- | The system is designed so it is possible to assemble BioBricks from the categories promotor, a gene of interest (GOI), a targeting signal (TS), a terminator, a marker cassette and a vector backbone. The marker cassette should consist of a promoter, a resistance gene, and a terminator. Ribosome binding site is integrated in the primer of the promoter when amplifying by PCR, which was the case for constructing the BioBrick and devices for the iGEM team <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Copenhagen"> Copenhagen</a>. | + | The system was designed based on the most common categories of biological part in the order; promotor, gene of interest (GOI), tail sequence (TS), terminator, marker cassette, and vector backbone. The standard marker cassette consists of a promoter, a gene, and a terminator. Short sequences are such as ribosomal binding sites (RBS) are meant to be integrated in the forward or reverse primer of the desired part when amplifying it by PCR. An example of this can be found on <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Copenhagen"> this page</a>. </p> |
- | | + | |
- | , the first thing we considered was which types of biobricks a device or other construct would consist of. We decided that the most systems would at least consist of a vector backbone, promoter, a gene of interest (GOI), a terminater, and a marker cassette.</p>
| + | |
| <br> | | <br> |
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| <p align="justify"> | | <p align="justify"> |
- | For all these parts to be assembled by USER cloning it was important that the overhangs (tails) of the different parts were not identical. Designing a different tail for each end of a part was important to ensure directionality and correct order of the biobricks. </p>
| + | The system is dependent on the assembly of the custom-made linkers on each part. The parts with linkers are synthesized by PCR amplification of template DNA using primers designed with an upstream extension of 8-9 additional nucleotides with a single deoxyuridine residue directly upstream the part. In order to assemble multiple parts while having a flexible system the linkers have an adenine in the 5' end and a thymine in the 3' end. |
- | | + | Furthermore, it is important that the linkers of the different parts are not identical to ensure the directionality and correct order of the biobricks.<br><br> |
- | <br> | + | Illustrated below, are the sequences of linkers between the parts, which are generated from the overhangs (linkers) on the PCR products. |
| + | </p> |
| <br> | | <br> |
- | <br>
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- |
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| <CENTER><img src="https://static.igem.org/mediawiki/2011/f/fe/Overveiw_tails_2.png" height="100px"> </img></center> | | <CENTER><img src="https://static.igem.org/mediawiki/2011/f/fe/Overveiw_tails_2.png" height="100px"> </img></center> |
- |
| |
| <br> | | <br> |
| + | <center><img src="https://static.igem.org/mediawiki/2011/2/27/Overveiw_module_tails-TS.png" height="100px" align="center"> </img></center> |
| <br> | | <br> |
| + | <p align="justify"> |
| + | The linkers are responsible for the flexibility of the system in that it for instance is possible use any promoter, since they will always have the same linkers. In the Plug 'n' Play system the coding sequence(s) can either consist of one gene of interest (GOI), called a module, or a GOI and a tail sequence, which could be any given DNA sequence of interest. </p> |
| <br> | | <br> |
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- | <center><img src="https://static.igem.org/mediawiki/2011/2/27/Overveiw_module_tails-TS.png" height="100px" align="center"> </img></center>
| |
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- | <br> | + | <a name="References"></a><h1><b>References</b></h1> |
- | <br>
| + | |
- | <br>
| + | |
- | <br>
| + | |
- | <font color="#990000" face="arial" size="5" >
| + | |
- | <p align="left"> | + | |
- | <br> | + | |
- | <b></b>References<br> | + | |
- | </p> | + | |
- | </font>
| + | |
| <p align="justify"> | | <p align="justify"> |
- | <br>
| + | (1) Hansen, B.G.; Salomonsen, B.; Nielsen, M. T.; Nielsen, J. B.; Hansen, N. B.; Nielsen, K. F.; Regueira, T. B.; Nielsen, J.; Patil, K. R.; Mortensen, U. F.; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.</p> |
- | (1) Hansen, Bjarke G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H. Mortensen; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.<br><br> | + | |
| | | |
- | (2) Bjarne Gram Hansen, Dorte Koefoed Holm, Morten Thrane Nielsen and Uffe Hasbro Mortensen. PCR based USER cloning for restriction enzyme and ligase-independent vector construction. Manuscript.<br><br> | + | <p align="justify"> |
| + | (2) Hansen, B. G.; Holm, D. K.; Nielsen M.T.; Mortensen, U.H. PCR based USER cloning for restriction enzyme and ligase-independent vector construction. Manuscript.</p> |
| | | |
- | (3) Rasmus JN Frandsen, Jens A Andersson, Matilde B Kristensen and Henriette Giese. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi.
BMC Molecular Biology 2008, 9:70.<br><br> | + | <p align="justify"> |
| + | (3) Frandsen, R. J. N.; Andersson, J.A.; Kristensen, M. B.; Giese, H. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi.
BMC Molecular Biology 2008, 9:70.</p> |
| | | |
- | (4) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).<br><br> | + | <p align="justify"> |
| + | (4) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).</p><br> |
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Plug 'n' Play with DNA assembly standard
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|
Making molecular biology easier
We imagine that iGEM should be about fast assembly of BioBricks, leaving more time for the actual project. It should be easy to combine any thinkable part, device or existing BioBrick. Unfortunately, classical cloning techniques can cause problems and even PCR can be cumbersome, if you have little or no laboratory experience.
Therefore, we introduce a simple and fast way of building new devices and vectors with ready to use PCR products. All that is needed is to select the favorite BioBricks and one of the ready to use backbone vectors, and mix it with USER enzyme. The assembled vector is ready for chemical transformation into competent E. coli cells only 70 minutes later.The 8-9 nucleotide overhangs (linkers) flanking the parts, can hybridize to complementary overhangs without the use of DNA-ligases.
No need to worry whether the destination vector has been fully linearized, no need to perform site-directed mutagenesis to remove unwished restriction recognition sites - just simple, fast, and easy.
It is possible to successfully assemble six biological parts in one reaction, reducing the plasmid construction time significantly. The upper limit of fragments that efficiently can be assembled has not yet been delineated (1).
In 2009 the first team from DTU participated in iGEM and introduced the USER fusion Assembly standard [BBF RFC 39]. This year we introduce a standardized version allowing easier use of the system. The Plug 'n' Play assembly standard can be found here (BBF RFC 80).
How it works
We have demonstrated that successful assembly of up to six biological parts in one reaction is possible e.g. (BBa_K678068).
The entire process of combining six BioBricks is illustrated in the figure below. The number of BioBricks can be adjusted as preferred and for instance mutations can easily be introduced by customizing the standard.
The procedure of assembly
Please refer to our Plug 'n' Play with DNA assembly standard BBF RFC 80 for a detailed protocol.
Pre-produced PCR products are selected from the Plug 'n' Play kit.
USER enzyme mix and buffer is added. This will remove the uracils, resulting in sticky ends on all BioBricks. The BioBricks and the backbone plasmid have matching sticky ends, and will self-assemble.
Chemical transformation of E. coli. The time of the final incubation of the transformation mix can be reduced to 30 min when using ampicillin as antibiotic resistance marker and still results in a high number of transformants.
If a part is not included in the Plug 'n' Play kit a simple PCR amplification can be performed on the part intended for the assembly in accordance to the Customization.
Plug 'n' Play benefits
Improvements of BBF RFC 39
No need to use a USER cassette, thereby completely avoiding the use of restriction enzymes and increasing the flexibility of the system.
Standardized linkers
What you get
Rapid assembly of biological parts
A well documented system
Assembly of multiple biobricks in one step
No illegal restriction sites
100% restriction enzyme free
100% DNA ligase free
High fidelity ensured by PfuTurbo® 7x Hotstart DNA polymerase
Directionality of inserts are supported
Any vector can be made Plug’n’Play compatible
Suitable for large scale high throughput projects
Vectors for Plug 'n' Play have been prepared by PCR ensuring 100% linear fragments. This means that the occurrence of false positives after bacterial transformation can be completely avoided (2).
The USER compatible backbone vectors were made by PCR amplification with primers containing the standardized linkers 6 and 1.
For all the PCR reactions it is important to remove the template vector before utilizing it in the USER reaction to avoid inauspicious transformations and false positives. The template can be removed by DpnI treatment or by gel band purification(2).
Previous studies have demonstrated a high level of correct assembly and directionality of fragments (2,3) and although we did not make such analysis ourselves it was in general our perception that this was the case, as of the more than 25 different Plug 'n' Play assemblies performed we encountered no more than two false-positives.
It is well known that mutations can be introduced during PCR. To minimize mutations the x7 proof-reading polymerase (4) that are fully compatible with the Plug 'n' Play standard should be used. The x7 proof-reading polymerase helps lowering the risk of introducing mutations.
The design of the system
The system was designed based on the most common categories of biological part in the order; promotor, gene of interest (GOI), tail sequence (TS), terminator, marker cassette, and vector backbone. The standard marker cassette consists of a promoter, a gene, and a terminator. Short sequences are such as ribosomal binding sites (RBS) are meant to be integrated in the forward or reverse primer of the desired part when amplifying it by PCR. An example of this can be found on this page.
The system is dependent on the assembly of the custom-made linkers on each part. The parts with linkers are synthesized by PCR amplification of template DNA using primers designed with an upstream extension of 8-9 additional nucleotides with a single deoxyuridine residue directly upstream the part. In order to assemble multiple parts while having a flexible system the linkers have an adenine in the 5' end and a thymine in the 3' end.
Furthermore, it is important that the linkers of the different parts are not identical to ensure the directionality and correct order of the biobricks.
Illustrated below, are the sequences of linkers between the parts, which are generated from the overhangs (linkers) on the PCR products.
The linkers are responsible for the flexibility of the system in that it for instance is possible use any promoter, since they will always have the same linkers. In the Plug 'n' Play system the coding sequence(s) can either consist of one gene of interest (GOI), called a module, or a GOI and a tail sequence, which could be any given DNA sequence of interest.
References
(1) Hansen, B.G.; Salomonsen, B.; Nielsen, M. T.; Nielsen, J. B.; Hansen, N. B.; Nielsen, K. F.; Regueira, T. B.; Nielsen, J.; Patil, K. R.; Mortensen, U. F.; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.
(2) Hansen, B. G.; Holm, D. K.; Nielsen M.T.; Mortensen, U.H. PCR based USER cloning for restriction enzyme and ligase-independent vector construction. Manuscript.
(3) Frandsen, R. J. N.; Andersson, J.A.; Kristensen, M. B.; Giese, H. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi.
BMC Molecular Biology 2008, 9:70.
(4) Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).
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